Parasitic helminth cuticlin proteins and uses thereof

ABSTRACT

The present invention relates to: parasitic helminth cuticlin proteins; parasitic helminth cuticlin nucleic acid molecules, including those that encode such cuticlin proteins; antibodies raised against such cuticlin proteins; and compounds that inhibit parasitic helminth cuticlin activity. The present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies, and inhibitory compounds. Also included in the present invention are therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies and/or inhibitory compounds as well as the use of such therapeutic compositions to protect animals from diseases caused by parasitic helminths.

FIELD OF THE INVENTION

The present invention relates to parasitic helminth cuticlin nucleicacid molecules, proteins encoded by such nucleic acid molecules,antibodies raised against such proteins, and inhibitors of suchproteins. The present invention also includes therapeutic compositionscomprising such nucleic acid molecules, proteins, antibodies,inhibitors, and combinations thereof, as well as the use of thesecompositions to protect animals from diseases caused by parasitichelminths, such as heartworm disease.

BACKGROUND OF THE INVENTION

Parasitic helminth infections in animals, including humans, aretypically treated by chemical drugs. One disadvantage with chemicaldrugs is that they must be administered often. For example, dogssusceptible to heartworm are typically treated monthly. Repeatedadministration of drugs, however, often leads to the development ofresistant helminth strains that no longer respond to treatment.Furthermore, many of the chemical drugs cause harmful side effects inthe animals being treated, and as larger doses become required due tothe build up of resistance, the side effects become even greater.Moreover, a number of drugs only treat symptoms of a parasitic diseasebut are unable to prevent infection by the parasitic helminth.

An alternative method to prevent parasitic helminth infection includesadministering a vaccine against a parasitic helminth. Although manyinvestigators have tried to develop vaccines based on specific antigens,it is well understood that the ability of an antigen to stimulateantibody production does not necessarily correlate with the ability ofthe antigen to stimulate an immune response capable of protecting ananimal from infection, particularly in the case of parasitic helminths.Although a number of prominent antigens have been identified in severalparasitic helminths, there is yet to be a commercially available vaccinedeveloped for any parasitic helminth.

As an example of the complexity of parasitic helminths, the life cycleof D. immitis, the helminth that causes heartworm disease, includes avariety of life forms, each of which presents different targets, andchallenges, for immunization. In a mosquito, D. immitis microfilariae gothrough two larval stages (L1 and L2) and become mature third stagelarvae (L3), which can then be transmitted back to the dog when themosquito takes a blood meal. In a dog, the L3 molt to the fourth larvalstage (L4), and subsequently to the fifth stage, or immature adults. Theimmature adults migrate to the heart and pulmonary arteries, where theymature to adult heartworms. Adult heartworms are quite large andpreferentially inhabit the heart and pulmonary arteries of an animal.Sexually mature adults, after mating, produce microfilariae whichtraverse capillary beds and circulate in the vascular system of the dog.

In particular, heartworm disease is a major problem in dogs, whichtypically do not develop immunity, even upon infection (i.e., dogs canbecome reinfected even after being cured by chemotherapy). In addition,heartworm disease is becoming increasingly widespread in other companionanimals, such as cats and ferrets. D. immitis has also been reported toinfect humans. There remains a need to identify an efficaciouscomposition that protects animals and humans against diseases caused byparasitic helminths, such as heartworm disease. Preferably, such acomposition also protects animals from infection by such helminths.

The cuticle is an important part of the nematode's exoskeleton andprotects the animal from the environment under a variety of conditions.In addition, it also mediates the metabolic interaction of the animalwith its environment and, in parasitic nematodes, the interaction withthe host and its immune system. The nematode cuticle is a complexextracellular structure that is secreted by an underlying syncytium ofhypodermal cells. Recent studies have demonstrated that the cuticle ofparasitic nematodes is a dynamic structure with important absorptive,secretory, and enzymatic activities, and not merely an inert protectivecovering as was once believed. See, for example, Lustigman, S. 1993,Parasitology Today, 9:8, 294-297. In addition, immunological studieshave shown the central importance of cuticular antigens as targets forprotective immune responses to parasitic nematodes. In spite of the widerecognition of the importance of the cuticle in the nematode physiologyand its potential role as a target for immunoprophylaxis, relativelylittle is known about the biology of the cuticle of filarial parasites.Though a number of collagen genes have been characterized in filarialparasites, very little is known about the non-collagenous cuticularproteins, including cuticlin, in filarial parasites. Prior studies in C.elegans have shown that cuticlin genes are developmentally regulated andthat the message for one of the C. elegans cuticlins, cut-1, isup-regulated during larval molting. Antibodies raised against a cuticlinof Ascaris cross-react with the epicuticular structures of filarialparasites indicating that components of cuticlin are immunogenic. Sincecuticlin proteins are highly conserved among nematodes, but not amongother organisms, they could be an important target for protectiveimmunity to parasitic helminths.

SUMMARY OF THE INVENTION

The present invention is based on the isolation of two D. immitisnucleic acid molecule isoforms, each encoding a protein with amino acidsequence similarity to cuticlin cut-1 proteins from C. elegans andAscaris lumbricodes.

The present invention relates to a novel product and process to protectanimals against parasitic helminth infection (e.g., to prevent and/ortreat such an infection). The present invention provides parasitichelminth cuticlin proteins and mimetopes thereof; parasitic helminthcuticlin nucleic acid molecules, including those that encode suchproteins; antibodies raised against such cuticlin proteins(anti-parasitic helminth cuticlin antibodies); and compounds thatinhibit cuticlin activity (i.e., inhibitory compounds or inhibitors).

The present invention also includes methods to obtain parasitic helminthcuticlin proteins, nucleic acid molecules, antibodies and inhibitorycompounds. Also included in the present invention are therapeuticcompositions comprising such proteins, nucleic acid molecules,antibodies, and inhibitory compounds, as well as use of such therapeuticcompositions to protect animals from diseases caused by parasitichelminths.

One embodiment of the present invention is an isolated nucleic acidmolecule that hybridizes under stringent hybridization conditions with aDirofilaria immitis (D. immitis) or Brugia malayi (B. malayi) cuticlingene. Such nucleic acid molecules are referred to as cuticlin nucleicacid molecules. A preferred isolated nucleic acid molecule of thisembodiment includes a D. immitis or B. malayi cuticlin nucleic acidmolecule. A D. immitis cuticlin nucleic acid molecule preferablyincludes nucleic acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:10, orallelic variants of any of these sequences. A B. malayi cuticlin nucleicacid molecule preferably includes nucleic acid sequence SEQ ID NO:16, orSEQ ID NO:18, or allelic variants of these sequences.

Another embodiment of the present invention is an isolated nucleic acidmolecule that includes a parasitic helminth cuticlin nucleic acidmolecule. A preferred parasitic helminth cuticlin nucleic acid moleculeof the present invention preferably includes nucleic acid sequence SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, or SEQ ID NO:18 orallelic variants of any of these sequences.

The present invention also relates to recombinant molecules, recombinantviruses and recombinant cells that include an isolated cuticlin nucleicacid molecule of the present invention. Also included are methods toproduce such nucleic acid molecules, recombinant molecules, recombinantviruses and recombinant cells.

Another embodiment of the present invention includes a non-nativeparasitic helminth cuticlin protein encoded by a nucleic acid moleculethat hybridizes under stringent hybridization conditions with aparasitic helminth cuticlin gene. A preferred parasitic helminth proteinis capable of eliciting an immune response when administered to ananimal and/or of having parasitic helminth cuticlin activity. Apreferred parasitic helminth cuticlin protein is encoded by a nucleicacid molecule that hybridizes under stringent conditions with a nucleicacid molecule including either SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:16, or SEQ ID NO:18, or allelic variants of any of these sequences.

Another embodiment of the present invention includes a parasitichelminth cuticlin protein. A preferred cuticlin protein includes a D.immitis or B. malayi cuticlin protein. A preferred D. immitis cuticlinprotein comprises amino acid sequence SEQ ID NO:4 or SEQ ID NO:9. Apreferred B. malayi cuticlin protein comprises amino acid sequence SEQID NO:17.

The present invention also relates to: mimetopes of parasitic helminthcuticlin proteins; isolated antibodies that selectively bind toparasitic helminth cuticlin proteins or mimetopes thereof; andinhibitors of parasitic helminth cuticlin proteins or mimetopes thereof.Also included are methods, including recombinant methods, to produceproteins, mimetopes, antibodies, and inhibitors of the presentinvention.

Another embodiment of the present invention is a method to identify acompound capable of inhibiting parasitic helminth cuticlin activity,comprising the steps of: (a) contacting a parasitic helminth cuticlinprotein with a putative inhibitory compound under conditions in which,in the absence of the compound, the protein has cuticlin activity; and(b) determining if the putative inhibitory compound inhibits thecuticlin activity. Also included in the present invention is a test kitto identify a compound capable of inhibiting parasitic helminth cuticlinactivity. Such a test kit includes a parasitic helminth cuticlin proteinhaving cuticlin activity and a means for determining the extent ofinhibition of the cuticlin activity in the presence of a putativeinhibitory compound.

Yet another embodiment of the present invention is a therapeuticcomposition that is capable of protecting an animal from disease causedby a parasitic helminth. Such a therapeutic composition includes one ormore of the following protective compounds: an isolated parasitichelminth cuticlin protein or a mimetope thereof; an isolated nucleicacid molecule that hybridizes under stringent hybridization conditionswith a Dirofilaria immitis cuticlin gene; an isolated antibody thatselectively binds to a parasitic helminth cuticlin protein; or aninhibitor of cuticlin protein activity identified by its ability toinhibit parasitic helminth cuticlin activity. A preferred therapeuticcomposition of the present invention also includes an excipient, anadjuvant, or a carrier. Preferred cuticlin nucleic acid moleculetherapeutic compositions of the present invention include geneticvaccines, recombinant virus vaccines, and recombinant cell vaccines.Also included in the present invention is a method to protect an animalfrom disease caused by a parasitic helminth, comprising the step ofadministering to the animal a therapeutic composition of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for isolated parasitic helminth cuticlinproteins, isolated parasitic helminth cuticlin nucleic acid molecules,isolated antibodies directed against parasitic helminth cuticlinproteins, and other inhibitors of parasitic helminth cuticlin activity.As used herein, the terms isolated parasitic helminth cuticlin proteins,and isolated parasitic helminth cuticlin nucleic acid molecules refersto cuticlin proteins and cuticlin nucleic acid molecules derived from aparasitic helminths and which can be obtained from their natural source,or can be produced using, for example, recombinant nucleic acidtechnology or chemical synthesis. Also included in the present inventionis the use of these proteins, nucleic acid molecules, antibodies andother inhibitors as therapeutic compositions to protect animals fromparasitic helminth diseases as well as in other applications, such asthose disclosed below.

The present invention is based on the isolation of two cDNAs encodingcuticlin cut-1 like proteins from D. immitis, and the isolation of ahomolog of these cDNAs from B. malayi. Parasitic helminth cuticlinproteins and nucleic acid molecules of the present invention haveutility because they represent novel targets for anti-parasite vaccinesand drugs. The products and processes of the present invention areadvantageous because they enable the inhibition of parasitephysiological functions that depend on cuticlin activity.

One embodiment of the present invention is an isolated proteincomprising a parasitic helminth cuticlin protein. It is to be noted thatthe term “a” or “an” entity refers to one or more of that entity; forexample, a protein refers to one or more proteins or at least oneprotein. As such, the terms “a” (or “an”), “one or more” and “at leastone” can be used interchangeably herein. It is also to be noted that theterms “comprising”, “including”, and “having” can be usedinterchangeably. Furthermore, a compound “selected from the groupconsisting of” refers to one or more of the compounds in the list thatfollows, including mixtures (i.e., combinations) of two or more of thecompounds. According to the present invention, an isolated, orbiologically pure, protein, is a protein that has been removed from itsnatural milieu. The terms “isolated” and “biologically pure” do notnecessarily reflect the extent to which the protein has been purified.An isolated protein of the present invention can be obtained from itsnatural source, can be produced using recombinant DNA technology or canbe produced by chemical synthesis. When an isolated protein of thepresent invention is produced using recombinant DNA technology orproduced by chemical synthesis, the protein is referred to herein aseither an isolated protein or as a non-native protein.

As used herein, an isolated parasitic helminth cuticlin protein can be afull-length protein or any homolog of such a protein. An isolatedprotein of the present invention, including a homolog, can be identifiedin a straight-forward manner by the protein's ability to elicit animmune response against a parasitic helminth cuticlin protein or tocatalyze the cleavage of asparagine to aspartic acid and ammonia.Examples of parasitic helminth cuticlin homologs include parasitichelminth cuticlin proteins in which amino acids have been deleted (e.g.,a truncated version of the protein, such as a peptide), inserted,inverted, substituted and/or derivatized (e.g., by glycosylation,phosphorylation, acetylation, myristoylation, prenylation,palmitoylation, amidation, or addition of glycerophosphatidyl inositol)so that the homolog includes at least one epitope capable of elicitingan immune response against a parasitic helminth cuticlin protein. Thatis, when the homolog is administered to an animal as an immunogen, usingtechniques known to those skilled in the art, the animal will produce animmune response against at least one epitope of a natural parasitichelminth cuticlin protein. As used herein, the term “epitope” refers tothe smallest portion of a protein or other antigen capable ofselectively binding to the antigen binding site of an antibody or aT-cell receptor. It is well accepted by those skilled in the art thatthe minimal size of a protein epitope is about four amino acids. Theability of a protein to effect an immune response can be measured usingtechniques known to those skilled in the art.

Parasitic helminth cuticlin protein homologs can be the result ofnatural allelic variation or natural mutation. Parasitic helminthcuticlin protein homologs of the present invention can also be producedusing techniques known in the art including, but not limited to, directmodifications to the protein or modifications to the gene encoding theprotein using, for example, classic or recombinant DNA techniques toeffect random or targeted mutagenesis.

A cuticlin protein of the present invention is encoded by a parasitichelminth cuticlin nucleic acid molecule. As used herein, a parasitichelminth cuticlin nucleic acid molecule includes a nucleic acid sequencerelated to a natural parasitic helminth cuticlin gene, and preferably,to a D. immitis or B. malayi cuticlin gene. As used herein, a parasitichelminth cuticlin gene includes all regions that control production ofthe parasitic helminth cuticlin protein encoded by the gene (such as,but not limited to, transcription, translation or post-translationcontrol regions) as well as the coding region itself, and any introns ornon-translated coding regions. As used herein, a gene that “includes” or“comprises” a nucleic acid sequence may include that sequence in onecontiguous array, or may include that sequence as fragmented exons. Asused herein, the term “coding region” refers to a continuous lineararray of nucleotides that translates into a protein. A full-lengthcoding region is that coding region which is translated into afull-length, i.e., a complete, protein as would be initially translatedin its natural milieu, prior to any post-translational modifications.

In one embodiment, a parasitic helminth cuticlin gene of the presentinvention includes the nucleic acid molecule represented by the nucleicacid sequence SEQ ID NO:1 (the coding strand), as well as the complementof SEQ ID NO:1. The production of this molecule (also referred to hereinas nDiCut-1A) is disclosed in the Examples. The complement of SEQ IDNO:1 (represented herein by SEQ ID NO:2) refers to the nucleic acidsequence of the strand complementary to the strand having SEQ ID NO:1,which can easily be determined by those skilled in the art. Likewise, anucleic acid sequence complement of any nucleic acid sequence of thepresent invention refers to the nucleic acid sequence of the nucleicacid strand that is complementary to (i.e., can form a double helixwith) the strand for which the sequence is cited.

In another embodiment, a parasitic helminth cuticlin gene of the presentinvention includes the nucleic acid sequence SEQ ID NO:6, as well as thecomplement of SEQ ID NO:6. Nucleic acid sequence SEQ ID NO:6 representsthe nucleic acid sequence of the coding strand of the nucleic acidmolecule denoted herein as nDiCut-1B, the production of which isdisclosed in the Examples. The complement of SEQ ID NO:6 (representedherein by SEQ ID NO:7) refers to the nucleic acid sequence of the strandcomplementary to the strand having SEQ ID NO:6.

In another embodiment, a parasitic helminth cuticlin gene of the presentinvention includes the nucleic acid sequence SEQ ID NO:16, as well asthe complement of SEQ ID NO:16. Nucleic acid sequence SEQ ID NO:16represents the nucleic acid sequence of the coding strand of the nucleicacid molecule denoted herein as BmCut-1A, the production of which isdisclosed in the Examples. The complement of SEQ ID NO:16 (representedherein by SEQ ID NO:18) refers to the nucleic acid sequence of thestrand complementary to the strand having SEQ ID NO:16.

In another embodiment, a parasitic helminth cuticlin gene can be anallelic variant that includes a similar, but not identical, sequence toSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, or SEQ ID NO:18. Forexample, an allelic variant of a parasitic helminth cuticlin geneincluding SEQ ID NO:1 and SEQ ID NO:2 is a gene that occurs atessentially the same locus (or loci) in the genome as the gene includingSEQ ID NO:1 and SEQ ID NO:2, but which, due to natural variations causedby, for example, mutation or recombination, has a similar but notidentical sequence. Because natural selection typically selects againstalterations that affect function, an allelic variant usually encodes aprotein having a similar activity or function to that of the proteinencoded by the gene to which it is being compared. An allelic variant ofa gene or nucleic acid molecule can also comprise alterations in the 5′or 3′ untranslated regions of the gene (e.g., in regulatory controlregions), or can involve alternative splicing of a nascent transcript,thereby bringing alternative exons into juxtaposition. Allelic variantsare well known to those skilled in the art and would be expected to befound naturally occurring within parasitic helminths because thehelminth genome is diploid, and sexual reproduction will result in thereassortment of alleles.

In one embodiment of the present invention, an isolated cuticlin proteinis encoded by a nucleic acid molecule that hybridizes under stringenthybridization conditions to a gene encoding a parasitic helminthcuticlin protein (i.e., to a D. immitis or B. malayi cuticlin gene). Theminimal size of a cuticlin protein of the present invention is a sizesufficient to be encoded by a nucleic acid molecule capable of forming astable hybrid (i.e., hybridize under stringent hybridization conditions)with the complementary sequence of a nucleic acid molecule encoding thecorresponding natural protein. The size of a nucleic acid moleculeencoding such a protein is dependent on the nucleic acid composition andthe percent homology between the parasitic helminth cuticlin nucleicacid molecule and the complementary nucleic acid sequence. It can easilybe understood that the extent of homology required to form a stablehybrid under stringent conditions can vary depending on whether thehomologous sequences are interspersed throughout a given nucleic acidmolecule or are clustered (i.e., localized) in distinct regions on agiven nucleic acid molecule.

The minimal size of a nucleic acid molecule capable of forming a stablehybrid with a gene encoding a parasitic helminth cuticlin protein istypically at least about 12 to about 15 nucleotides in length if thenucleic acid molecule is GC-rich and at least about 15 to about 17nucleotides in length if it is AT-rich. The minimal size of a nucleicacid molecule used to encode a cuticlin protein homolog of the presentinvention is from about 12 to about 18 nucleotides in length. Thus, theminimal size of a cuticlin protein homolog of the present invention isfrom about 4 to about 6 amino acids in length. There is no limit, otherthan a practical limit, on the maximal size of a nucleic acid moleculeencoding a parasitic helminth cuticlin protein or protein homologbecause a nucleic acid molecule of the present invention can include aportion of a gene, an entire gene, or multiple genes. The preferred sizeof a protein encoded by a nucleic acid molecule of the present inventiondepends on whether a full-length, fusion, multivalent, or functionalportion of such a protein is desired.

Stringent hybridization conditions are determined based on definedphysical properties of the gene to which the nucleic acid molecule isbeing hybridized, and can be defined mathematically. Stringenthybridization conditions are those experimental parameters that allow anindividual skilled in the art to identify significant similaritiesbetween heterologous nucleic acid molecules. These conditions are wellknown to those skilled in the art. See, for example, Sambrook, et al,1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LabsPress, and Meinkoth, et al., 1984, Anal. Biochem. 138, 267-284, each ofwhich is incorporated by reference herein in its entirety. As explainedin detail in the cited references, the determination of hybridizationconditions involves the manipulation of a set of variables including theionic strength (M, in moles/liter), the hybridization temperature (°C.), the concentration of nucleic acid helix destabilizing agents (suchas formamide), the average length of the shortest hybrid duplex (n), andthe percent G+C composition of the fragment to which an unknown nucleicacid molecule is being hybridized. For nucleic acid molecules of atleast about 150 nucleotides, these variables are inserted into astandard mathematical formula to calculate the melting temperature, orT_(m), of a given nucleic acid molecule. As defined in the formulabelow, T_(m) is the temperature at which two complementary nucleic acidmolecule strands will disassociate, assuming 100% complementaritybetween the two strands:

T _(m)=81.5° C.+16.6 log M+0.41 (% G+C)−500/n−0.61 (% formamide).

For nucleic acid molecules smaller than about 50 nucleotides, hybridstability is defined by the dissociation temperature (T_(d)), which isdefined as the temperature at which 50% of the duplexes dissociate. Forthese smaller molecules, the stability at a standard ionic strength isdefined by the following equation:

T _(d)=4(G+C)+2(A+T).

A temperature of 5° C. below T_(d) is used to detect hybridizationbetween perfectly matched molecules.

Also well known to those skilled in the art is how base pair mismatch,i.e. differences between two nucleic acid molecules being compared,including non-complementarity of bases at a given location, and gaps dueto insertion or deletion of one or more bases at a given location oneither of the nucleic acid molecules being compared, will affect T_(m)or T_(d) for nucleic acid molecules of different sizes. For example,T_(m) decreases about 1° C. for each 1% of mismatched base pairs forhybrids greater than about 150 bp, and T_(d) decreases about 5° C. foreach mismatched base pair for hybrids below about 50 bp. Conditions forhybrids between about 50 and about 150 base pairs can be determinedempirically and without undue experimentation using standard laboratoryprocedures well known to those skilled in the art. These simpleprocedures allow one skilled in the art to set the hybridizationconditions (by altering, for example, the salt concentration, theformamide concentration or the temperature) so that only nucleic acidhybrids with greater than a specified % base pair mismatch willhybridize. Stringent hybridization conditions are commonly understood bythose skilled in the art to be those experimental conditions that willallow less than or equal to about 30% base pair mismatch (i.e., at leastabout 70% identity). Because one skilled in the art can easily determinewhether a given nucleic acid molecule to be tested is less than orgreater than about 50 nucleotides, and can therefore choose theappropriate formula for determining hybridization conditions, he or shecan determine whether the nucleic acid molecule will hybridize with agiven gene under stringent hybridization conditions and similarlywhether the nucleic acid molecule will hybridize under conditionsdesigned to allow a desired amount of base pair mismatch.

Hybridization reactions are often carried out by attaching the nucleicacid molecule to be hybridized to a solid support such as a membrane,and then hybridizing with a labeled nucleic acid molecule, typicallyreferred to as a probe, suspended in a hybridization solution. Examplesof common hybridization reaction techniques include, but are not limitedto, the well-known Southern and northern blotting procedures. Typically,the actual hybridization reaction is done under non-stringentconditions, i.e., at a lower temperature and/or a higher saltconcentration, and then high stringency is achieved by washing themembrane in a solution with a higher temperature and/or lower saltconcentration in order to achieve the desired stringency.

For example, if the skilled artisan wished to identify a nucleic acidmolecule that hybridizes under conditions that would allow less than orequal to 30% pair mismatch with a flea nucleic acid molecule of about150 bp in length or greater, the following conditions could preferablybe used. The average G+C content of D. immitis DNA is about 35%, ascalculated from known flea nucleic acid sequences. The unknown nucleicacid molecules would be attached to a support membrane, and the 150 bpprobe would be labeled, e.g. with a radioactive tag. The hybridizationreaction could be carried out in a solution comprising 2×SSC and 0% formamide, at a temperature of about 37° C. (low stringency conditions).Solutions of differing concentrations of SSC can be made by one of skillin the art by diluting a stock solution of 20×SSC (175.3 gram NaCl andabout 88.2 gram sodium citrate in 1 liter of water, pH 7) to obtain thedesired concentration of SSC. The skilled artisan would calculate thewashing conditions required to allow up to 30% base pair mismatch. Forexample, in a wash solution comprising 1×SSC and 0% formamide, the T_(m)of perfect hybrids would be about 79° C.:

81.5° C.+16.6 log (0.15M)+(0.41×0.35)−(500/150)−(0.61×0)=79° C.

Thus, to achieve hybridization with nucleic acid molecules having about30% base pair mismatch, hybridization washes would be carried out at atemperature of less than or equal to 49° C. It is thus within the skillof one in the art to calculate additional hybridization temperaturesbased on the desired percentage base pair mismatch, formulae and G/Ccontent disclosed herein. For example, it is appreciated by one skilledin the art that as the nucleic acid molecule to be tested forhybridization against nucleic acid molecules of the present inventionhaving sequences specified herein becomes longer than 150 nucleotides,the T_(m) for a hybridization reaction allowing up to 30% base pairmismatch will not vary significantly from 49° C.

Furthermore, it is known in the art that there are commerciallyavailable computer programs for determining the degree of similaritybetween two nucleic acid sequences. These computer programs includevarious known methods to determine the percentage identity and thenumber and length of gaps between hybrid nucleic acid molecules. It isfurther known that the various available sequence analysis programsproduce substantially similar results when the two compared moleculesencode amino acid sequences that have greater than 30% amino acididentity. See Johnson et al., J. Mol. Biol., vol. 233, pages 716-738,1993, and Feng et al., J. Mol. Evol., vol. 21, pages 112-125, 1985, bothof which are incorporated by reference herein in their entirety.Preferred methods to determine the percent identity among amino acidsequences and also among nucleic acid sequences include analysis usingone or more of the commercially available computer programs designed tocompare and analyze nucleic acid or amino acid sequences. These computerprograms include, but are in no way limited to, GCG™ (available fromGenetics Computer Group, Madison, Wis.), DNAsis™ (available from HitachiSoftware, San Bruno, Calif.) and MacVector™ (available from the EastmanKodak Company, New Haven, Conn.). A particularly preferred method todetermine the percent identity among amino acid sequences and also amongnucleic acid sequences is to perform the analysis using the DNAsis™computer program, using default parameters.

A preferred parasitic helminth cuticlin protein of the present inventionis a compound that when administered to an animal in an effectivemanner, is capable of protecting that animal from disease caused by aparasitic helminth. In accordance with the present invention, theability of a cuticlin protein of the present invention to protect ananimal from disease by a parasitic helminth refers to the ability ofthat protein to, for example, treat, ameliorate or prevent diseasecaused by parasitic helminths. In one embodiment, a parasitic helminthcuticlin protein of the present invention can elicit an immune response(including a humoral and/or cellular immune response) against aparasitic helminth.

Suitable parasites to target include any parasite that is essentiallyincapable of causing disease in an animal administered a parasitichelminth cuticlin protein of the present invention. Accordingly, aparasite to target includes any parasite that produces a protein havingone or more epitopes that can be targeted by a humoral or cellularimmune response against a parasitic helminth cuticlin protein of thepresent invention or that can be targeted by a compound that otherwiseinhibits parasite cuticlin activity, thereby resulting in the decreasedability of the parasite to cause disease in an animal. Preferredparasites to target include parasitic helminths such as nematodes,cestodes, and trematodes, with nematodes being preferred. Preferrednematodes to target include filariid, ascarid, capillarid, strongylid,strongyloides, trichostrongyle, and trichurid nematodes. Particularlypreferred nematodes are those of the genera Acanthocheilonema,Aelurostrongylus, Ancylostoma, Angiostrongylus, Ascaris, Brugia,Bunostomum, Capillaria, Chabertia, Cooperia, Crenosoma, Dictyocaulus,Dioctophyme, Dipetalonema, Diphyllobothrium, Diplydium, Dirofilaria,Dracunculus, Enterobius, Filaroides, Haemonchus, Lagochilascaris, Loa,Mansonella, Muellerius, Nanophyetus, Necator, Nematodirus,Oesophagostomum, Onchocerca, Opisthorchis, Ostertagia, Parafilaria,Paragonimus, Parascaris, Physaloptera, Protostrongylus, Setaria,Spirocerca, Spirometra, Stephanofilaria, Strongyloides, Strongylus,Thelazia, Toxascaris, Toxocara, Trichinella, Trichostrongylus,Trichuris. Uncinaria, and Wuchereria. Preferred filariid nematodesinclude Dirofilaria, Onchocerca, Acanthocheilonema, Brugia,Dipetalonema, Loa, Parafilaria, Setaria, Stephanofilaria and Wuchereriafilariid nematodes, with D. immitis being even more preferred.

The present invention also includes mimetopes of parasitic helminthcuticlin proteins of the present invention. As used herein, a mimetopeof a parasitic helminth cuticlin protein of the present invention refersto any compound that is able to mimic the activity of a parasitichelminth cuticlin protein (e.g., has the ability to elicit an immuneresponse against a parasitic helminth cuticlin protein of the presentinvention or ability to inhibit parasitic helminth cuticlin activity).The ability to mimic the activity of a parasitic helminth cuticlinprotein is likely to be the result of a structural similarity betweenthe parasitic helminth cuticlin protein and the mimetope. It is to benoted, however, that the mimetope need not have a structure similar to aparasitic helminth cuticlin protein as long as the mimetope functionallymimics the protein. A mimetope can be, but is not limited to: a peptidethat has been modified to decrease its susceptibility to degradation(e.g., as an all-D retro peptide); an anti-idiotypic or catalyticantibody, or a fragment thereof; a non-proteinaceous immunogenic portionof an isolated protein (e.g., a carbohydrate structure); or a syntheticor natural organic molecule, including a nucleic acid. Such a mimetopecan be designed using computer-generated structures of proteins of thepresent invention. A mimetope can also be obtained by generating randomsamples of molecules, such as oligonucleotides, peptides or otherorganic molecules, and screening such samples by affinity chromatographytechniques using the corresponding binding partner.

In one embodiment, a parasitic helminth cuticlin protein of the presentinvention is a fusion protein that includes a parasitic helminthcuticlin protein-containing domain attached to one or more fusionsegments. Suitable fusion segments for use with the present inventioninclude, but are not limited to, segments that can: enhance a protein'sstability; act as an immunopotentiator to enhance an immune responseagainst a parasitic helminth cuticlin protein; or assist purification ofa parasitic helminth cuticlin protein (e.g., by affinitychromatography). A suitable fusion segment can be a domain of any sizethat has the desired function (e.g., imparts increased stability,imparts increased immunogenicity to a protein, or simplifiespurification of a protein). Fusion segments can be joined to the aminoor carboxyl termini of a parasitic helminth cuticlin protein-containingdomain, and can be susceptible to cleavage in order to enablestraight-forward recovery of a parasitic helminth cuticlin protein. Afusion protein is preferably produced by culturing a recombinant celltransformed with a fusion nucleic acid molecule that encodes a proteinincluding a fusion segment attached to either the carboxyl or aminoterminal end of a cuticlin protein-containing domain. Preferred fusionsegments include a metal binding domain (e.g., a poly-histidinesegment); an immunoglobulin binding domain (e.g., Protein A; Protein G;T cell; B cell; Fc receptor or complement protein antibody-bindingdomains); a sugar binding domain (e.g., a maltose binding domain);and/or a “tag” domain (e.g., at least a portion of β-galactosidase, astrep tag peptide, a T7-tag peptide, a FLAG™ peptide, or other domainthat can be purified using compounds that bind to the domain, such asmonoclonal antibodies). More preferred fusion segments include metalbinding domains, such as a poly-histidine segment; a maltose bindingdomain; a strep tag peptide, such as that available from Biometra® inTampa, Fla.; and an S10 peptide.

In another embodiment, a parasitic helminth cuticlin protein of thepresent invention also includes at least one additional protein segmentthat is capable of protecting an animal from one or more diseases. Sucha multivalent protective protein can be produced by culturing a celltransformed with a nucleic acid molecule comprising two or more nucleicacid domains joined together in such a manner that the resulting nucleicacid molecule is expressed as a multivalent protective compoundcontaining at least two protective compounds, or portions thereof,capable of protecting an animal from diseases caused, for example, by atleast one infectious agent.

Examples of multivalent protective compounds include, but are notlimited to, a parasitic helminth cuticlin protein of the presentinvention attached to one or more compounds protective against one ormore other infectious agents, particularly an agent that infects humans,cats, dogs, ferrets, cattle or horses, such as, but not limited to:viruses (e.g., adenoviruses, caliciviruses, coronaviruses, distemperviruses, hepatitis viruses, herpesviruses, immunodeficiency viruses,infectious peritonitis viruses, leukemia viruses, oncogenic viruses,panleukopenia viruses, papilloma viruses, parainfluenza viruses,parvoviruses, rabies viruses, and reoviruses, as well as othercancer-causing or cancer-related viruses); bacteria (e.g., Actinomyces,Bacillus, Bacteroides, Bordetella, Bartonella, Borrelia, Brucella,Campylobacter, Capnocytophaga, Clostridium, Corynebacterium, Coxiella,Dermatophilus, Enterococcus, Ehrlichia, Escherichia, Francisella,Fusobacterium, Haemobartonella, Helicobacter, Klebsiella, L-formbacteria, Leptospira, Listeria, Mycobacteria, Mycoplasma, Neorickettsia,Nocardia, Pasteurella, Peptococcus, Peptostreptococcus, Proteus,Pseudomonas, Rickettsia, Rochalimaea, Salmonella, Shigella,Staphylococcus, Streptococcus, and Yersinia; fungi and fungal-relatedmicroorganisms (e.g., Absidia, Acremonium, Alternaria, Aspergillus,Basidiobolus, Bipolaris, Blastomyces, Candida, Chlamydia, Coccidioides,Conidiobolus, Cryptococcus, Curvalaria, Epidermophyton, Exophiala,Geotrichum, Histoplasma, Madurella, Malassezia, Microsporum, Moniliella,Mortierella, Mucor, Paecilomyces, Penicillium, Phialemonium,Phialophora, Prototheca, Pseudallescheria, Pseudomicrodochium, Pythium,Rhinosporidium, Rhizopus, Scolecobasidium, Sporothrix, Stemphylium,Trichophyton, Trichosporon, and Xylohypha; and other parasites (e.g.,Babesia, Balantidium, Besnoitia, Cryptosporidium, Eimeria,Encephalitozoon, Entamoeba, Giardia, Hammondia, Hepatozoon, Isospora,Leishmania, Microsporidia, Neospora, Nosema, Pentatrichomonas,Plasmodium, Pneumocystis, Sarcocystis, Schistosoma, Theileria,Toxoplasma, and Trypanosoma, as well as helminth parasites, such asthose disclosed herein). In one embodiment, a parasitic helminthcuticlin protein of the present invention is attached to one or moreadditional compounds protective against heartworm disease. In anotherembodiment, one or more protective compounds, such as those listedabove, can be included in a multivalent vaccine comprising a parasitichelminth cuticlin protein of the present invention and one or more otherprotective molecules as separate compounds.

In one embodiment, a preferred isolated cuticlin protein of the presentinvention is a protein encoded by a nucleic acid molecule comprising atleast a portion of nDiCut-1A, nDiCut-1B, or nBmCut-1A, or by an allelicvariant of any of these nucleic acid molecules. Also preferred is anisolated cuticlin protein encoded by a nucleic acid molecule having thenucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, SEQ IDNO:8, or SEQ ID NO:16; or by an allelic variant of a nucleic acidmolecule having any of these sequences.

Translation of SEQ ID NO:1, the coding strand of nucleic acid moleculenDiCut-1A, yields an essentially full length parasitic helminth cuticlinprotein of 387 amino acids, referred to herein as PDiCut-1A, the aminoacid sequence of which is represented by SEQ ID NO:4. The open readingframe spans from nucleotide 167 through nucleotide 1327 of SEQ ID NO:1and a termination (stop) codon spans from nucleotide 1329 throughnucleotide 1331 of SEQ ID NO:1. The coding region encoding PDiCut-1A, isrepresented by SEQ ID NO:3 (the coding strand) and SEQ ID NO:5 (thecomplementary strand).

Translation of SEQ ID NO:6, the coding strand of nucleic acid moleculenDiCut-1B, yields a full length parasitic helminth cuticlin protein of271 amino acids, referred to herein as PDiCut-1B, the amino acidsequence of which is represented by SEQ ID NO:9, assuming an openreading frame that spans from nucleotide 392 through nucleotide 1203 ofSEQ ID NO:6. The coding region encoding PDiCut-1B is represented by SEQID NO:8 (the coding strand) and SEQ ID NO:10 (the complementary strand).The deduced amino acid sequence is represented by SEQ ID NO:9.

Translation of SEQ ID NO:16, the coding strand of nucleic acid moleculenBmCut-1A, yields a partial length parasitic helminth cuticlin proteinof 245 amino acids, referred to herein as PBmCut-1A, the amino acidsequence of which is represented by SEQ ID NO:17. The open reading framespans from nucleotide 158 through nucleotide 892 of SEQ ID NO:16.

One embodiment of the present invention includes a non-native parasitichelminth cuticlin protein encoded by a nucleic acid molecule thathybridizes under stringent hybridization conditions with a parasitichelminth cuticlin gene. A preferred parasitic helminth cuticlin proteinis capable of eliciting an immune response when administered to ananimal and/or of having parasitic helminth cuticlin activity. Apreferred parasitic helminth cuticlin protein is encoded by a nucleicacid molecule that hybridizes under stringent conditions with a nucleicacid molecule including either SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:16, or SEQ ID NO:18, or with allelic variants of any of thesesequences

A preferred cuticlin protein includes a protein encoded by a nucleicacid molecule which is at least about 50 nucleotides and whichhybridizes under conditions which preferably allow about 20% base pairmismatch, more preferably under conditions which allow about 15% basepair mismatch, more preferably under conditions which allow about 10%base pair mismatch, more preferably under conditions which allow about5% base pair mismatch, and even more preferably under conditions whichallow about 2% base pair mismatch with a nucleic acid molecule selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:16, or SEQ ID NO:18.

Another preferred cuticlin protein of the present invention includes aprotein encoded by a nucleic acid molecule which is at least about 150nucleotides and which hybridizes under conditions which preferably allowabout 30% base pair mismatch, more preferably under conditions whichallow about 25% base pair mismatch, more preferably under conditionswhich allow about 20% base pair mismatch, more preferably underconditions which allow about 15% base pair mismatch, more preferablyunder conditions which allow about 10% base pair mismatch, morepreferably under conditions which allow about 5% base pair mismatch, andeven more preferably under conditions which allow about 2% base pairmismatch with a nucleic acid molecule selected from the group consistingof SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, or SEQ ID NO:18.

Another embodiment of the present invention includes a cuticlin proteinencoded by a nucleic acid molecule comprising at least about 50nucleotides, wherein said nucleic acid molecule hybridizes, in asolution comprising 2×SSC and 0% formamide, at a temperature of 37° C.,and washing in 1×SSC and 0% formamide at a temperature of 64° C., to anisolated nucleic acid molecule selected from the group consisting of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, or SEQ ID NO:18.Additional preferred cuticlin proteins include proteins encoded byoligonucleotides of an isolated nucleic acid molecule comprising atleast about 50 nucleotides, wherein said nucleic acid moleculehybridizes, in a solution comprising 2×SSC and 0% formamide, at atemperature of 37° C., and washing in 1×SSC and 0% formamide at atemperature of 64° C., to an isolated nucleic acid molecule selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:16, or SEQ ID NO:18, wherein said oligonucleotide comprises at leastabout 50 nucleotides.

Another embodiment of the present invention includes a cuticlin proteinencoded by a nucleic acid molecule comprising at least about 150nucleotides, wherein said nucleic acid molecule hybridizes, in asolution comprising 2×SSC and 0% formamide, at a temperature of 37° C.,and washing in 1×SSC and 0% formamide at a temperature of 64° C., to anisolated nucleic acid molecule selected from the group consisting of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, or SEQ ID NO:18.Additional preferred cuticlin proteins include proteins encoded byoligonucleotides of an isolated nucleic acid molecule comprising atleast about 150 nucleotides, wherein said nucleic acid moleculehybridizes, in a solution comprising 2×SSC and 0% formamide, at atemperature of 37° C., and washing in 1×SSC and 0% formamide at atemperature of 64° C., to an isolated nucleic acid molecule selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:16, or SEQ ID NO:18, wherein said oligonucleotide comprises at leastabout 50 nucleotides.

A preferred cuticlin protein of the present invention comprises aprotein that is that is at least about 75%, more preferably at leastabout 80%, more preferably at least about 85%, more preferably at leastabout 90%, more preferably at least about 95%, and more preferably atleast about 98% identical to identical to PDiCut-1A, PDiCut-1B, orPBmCut-1A. More preferred is a cuticlin protein comprising PDiCut-1A,PDiCut-1B, or PBmCut-1A, or a protein encoded by an allelic variant of anucleic acid molecule encoding a protein comprising PDiCut-1A,PDiCut-1B, or PBmCut-1A.

Also preferred is a cuticlin protein comprising an amino acid sequencethat is at least about 75%, more preferably at least about 80%, morepreferably at least about 85%, more preferably at least about 90%, morepreferably at least about 95%, and more preferably at least about 98%identical to amino acid sequence SEQ ID NO:4, SEQ ID NO:9, or SEQ IDNO:17. Even more preferred is an amino acid sequence having the sequencerepresented by SEQ ID NO:4, SEQ ID NO:9, or SEQ ID NO:17, or an allelicvariant of any of these an amino acid sequences.

In one embodiment, a preferred parasitic helminth cuticlin proteincomprises an amino acid sequence of at least about 5 amino acids,preferably at least about 10 amino acids, more preferably at least about15 amino acids, more preferably at least about 20 amino acids, morepreferably at least about 25 amino acids, more preferably at least about30 amino acids, more preferably at least about 35 amino acids, morepreferably at least about 50 amino acids, more preferably at least about100 amino acids, more preferably at least about 200 amino acids, morepreferably at least about 250 amino acids, more preferably at leastabout 275 amino acids, more preferably at least about 300 amino acids,more preferably at least about 350 amino acids, more preferably at leastabout 375 amino acids, and even more preferably at least about 400 aminoacids. In another embodiment, preferred parasitic helminth cuticlinproteins comprise full-length proteins, i.e., proteins encoded byfull-length coding regions, or post-translationally modified proteinsthereof, such as mature proteins from which initiating methionine and/orsignal sequences or “pro” sequences have been removed.

A fragment of a parasitic helminth cuticlin protein of the presentinvention preferably comprises at least about 5 amino acids, morepreferably at least about 10 amino acids, more preferably at least about15 amino acids, more preferably at least about 20 amino acids, morepreferably at least about 25 amino acids, more preferably at least about30 amino acids, more preferably at least about 35 amino acids, morepreferably at least about 40 amino acids, more preferably at least about45 amino acids, more preferably at least about 50 amino acids, morepreferably at least about 55 amino acids, more preferably at least about60 amino acids, more preferably at least about 65 amino acids, morepreferably at least about 70 amino acids, more preferably at least about75 amino acids, more preferably at least about 80 amino acids, morepreferably at least about 85 amino acids, more preferably at least about90 amino acids, more preferably at least about 95 amino acids, and evenmore preferably at least about 100 amino acids in length.

A particularly preferred parasitic helminth cuticlin protein of thepresent invention comprises amino acid sequence SEQ ID NO:4, including,but not limited to, a cuticlin protein consisting of amino acid sequenceSEQ ID NO:4, a fusion protein or a multivalent protein; or a proteinencoded by an allelic variant of a nucleic acid molecule encoding aprotein having amino acid sequence SEQ ID NO:4. Also particularlypreferred is a parasitic helminth cuticlin protein of the presentinvention that comprises amino acid sequence SEQ ID NO:9, including, butnot limited to, a cuticlin protein consisting of amino acid sequence SEQID NO:9, a fusion protein or a multivalent protein; or a protein encodedby an allelic variant of a nucleic acid molecule encoding a proteinhaving amino acid sequence SEQ ID NO:9. Also particularly preferred is aparasitic helminth cuticlin protein of the present invention thatcomprises amino acid sequence SEQ ID NO:17, including, but not limitedto, a cuticlin protein consisting of amino acid sequence SEQ ID NO:17, afusion protein or a multivalent protein; or a protein encoded by anallelic variant of a nucleic acid molecule encoding a protein havingamino acid sequence SEQ ID NO:17.

Another embodiment of the present invention is an isolated nucleic acidmolecule comprising a parasitic helminth cuticlin nucleic acid molecule.The identifying characteristics of such a nucleic acid molecule areheretofore described. A nucleic acid molecule of the present inventioncan include an isolated natural parasitic helminth cuticlin gene or ahomolog thereof, the latter of which is described in more detail below.A nucleic acid molecule of the present invention can include one or moreregulatory regions, a full-length or a partial coding region, or acombination thereof. The minimal size of a nucleic acid molecule of thepresent invention is a size sufficient to allow the formation of astable hybrid (i.e., hybridization under stringent hybridizationconditions) with the complementary sequence of another nucleic acidmolecule. Accordingly, the minimal size of a cuticlin nucleic acidmolecule of the present invention is from about 12 to about 18nucleotides in length. A preferred cuticlin nucleic acid moleculeincludes a parasitic helminth cuticlin nucleic acid molecule.

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subject to human manipulation) andcan include DNA, RNA, or derivatives of either DNA or RNA. As such,“isolated” does not reflect the extent to which the nucleic acidmolecule has been purified. An isolated parasitic helminth cuticlinnucleic acid molecule of the present invention can be isolated from itsnatural source or produced using recombinant DNA technology (e.g.,polymerase chain reaction (PCR) amplification or cloning) or chemicalsynthesis. Isolated parasitic helminth cuticlin nucleic acid moleculescan include, for example, natural allelic variants and nucleic acidmolecules modified by nucleotide insertions, deletions, substitutions,or inversions in a manner such that the modifications do notsubstantially interfere with the nucleic acid molecule's ability toencode a cuticlin protein of the present invention.

A parasitic helminth cuticlin nucleic acid molecule homolog can beproduced using a number of methods known to those skilled in the art.See, for example, Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Labs Press; Sambrook et al., ibid., isincorporated by reference herein in its entirety. For example, a nucleicacid molecule can be modified using a variety of techniques including,but not limited to, classic mutagenesis and recombinant DNA techniquessuch as site-directed mutagenesis, chemical treatment, restrictionenzyme cleavage, ligation of nucleic acid fragments, PCR amplification,synthesis of oligonucleotide mixtures and ligation of mixture groups to“build” a mixture of nucleic acid molecules, and combinations thereof. Anucleic acid molecule homolog can be selected by hybridization with aparasitic helminth cuticlin nucleic acid molecule or by screening thefunction of a protein encoded by the nucleic acid molecule (e.g.,ability to elicit an immune response against at least one epitope of aparasitic helminth cuticlin protein, or the ability to demonstratecuticlin activity).

An isolated nucleic acid molecule of the present invention can include anucleic acid sequence that encodes a parasitic helminth cuticlin proteinof the present invention, examples of such proteins being disclosedherein. Although the phrase “nucleic acid molecule” primarily refers tothe physical nucleic acid molecule and the phrase “nucleic acidsequence” primarily refers to the sequence of nucleotides on the nucleicacid molecule, the two phrases can be used interchangeably, especiallywith respect to a nucleic acid molecule, or a nucleic acid sequence,being capable of encoding a parasitic helminth cuticlin protein.

A preferred nucleic acid molecule of the present invention, whenadministered to an animal, is capable of protecting that animal fromdisease caused by a parasitic helminth. As will be disclosed in moredetail below, such a nucleic acid molecule can be, or can encode, anantisense RNA, a molecule capable of triple helix formation, a ribozyme,or other nucleic acid-based drug compound. In additional embodiments, anucleic acid molecule of the present invention can encode a protectiveprotein (e.g., a cuticlin protein of the present invention), the nucleicacid molecule being delivered to the animal, for example, by directinjection (i.e., as a genetic vaccine) or in a vehicle such as arecombinant virus vaccine or a recombinant cell vaccine.

One embodiment of the present invention is an isolated nucleic acidmolecule that hybridizes under stringent hybridization conditions with aparasitic helminth cuticlin gene. Preferred parasitic helminth cuticlingenes of the present invention are cuticlin genes from Dirofilariaimmitis or B. malayi. Such nucleic acid molecules are referred to asparasitic helminth cuticlin nucleic acid molecules. A parasitic helminthcuticlin gene preferably includes at least one of the following nucleicacid sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:16, or SEQ IDNO:18.

In one embodiment of the present invention, a preferred parasitichelminth cuticlin nucleic acid molecule includes an isolated nucleicacid molecule which is at least about 50 nucleotides and whichhybridizes under conditions which preferably allow about 20% base pairmismatch, more preferably under conditions which allow about 15% basepair mismatch, more preferably under conditions which allow about 10%base pair mismatch and even more preferably under conditions which allowabout 5% base pair mismatch with a nucleic acid molecule selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:16,or SEQ ID NO:18.

Another preferred parasitic helminth cuticlin nucleic acid molecule ofthe present invention includes a nucleic acid molecule which is at leastabout 150 nucleotides and which hybridizes under conditions whichpreferably allow about 30% base pair mismatch, more preferably underconditions which allow about 25% base pair mismatch, more preferablyunder conditions which allow about 20% base pair mismatch, morepreferably under conditions which allow about 15% base pair mismatch,more preferably under conditions which allow about 10% base pairmismatch and even more preferably under conditions which allow about 5%base pair mismatch with a nucleic acid molecule selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:16, or SEQ IDNO:18.

Another embodiment of the present invention includes a nucleic acidmolecule comprising at least about 50 nucleotides, wherein said nucleicacid molecule hybridizes, in a solution comprising 2×SSC and 0%formamide, at a temperature of 37° C., and washing in 1×SSC and 0%formamide at a temperature of 64° C., to an isolated nucleic acidmolecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:16, or SEQ ID NO:18. Additional preferred nucleic acidmolecules of the present invention include oligonucleotides of anisolated nucleic acid molecule comprising at least about 50 nucleotides,wherein said nucleic acid molecule hybridizes, in a solution comprising2×SSC and 0% formamide, at a temperature of 37° C., and washing in 1×SSCand 0% formamide at a temperature of 64° C., to an isolated nucleic acidmolecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:16, or SEQ ID NO:18, wherein said oligonucleotidecomprises at least about 50 nucleotides.

Another embodiment of the present invention includes a nucleic acidmolecule comprising at least about 150 nucleotides, wherein said nucleicacid molecule hybridizes, in a solution comprising 2×SSC and 0%formamide, at a temperature of 37° C., and washing in 1×SSC and 0%formamide at a temperature of 64° C., to an isolated nucleic acidmolecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:16, or SEQ ID NO:18. Additional preferred nucleic acidmolecules of the present invention include oligonucleotides of anisolated nucleic acid molecule comprising at least about 150nucleotides, wherein said nucleic acid molecule hybridizes, in asolution comprising 2×SSC and 0% formamide, at a temperature of 37° C.,and washing in 1×SSC and 0% formamide at a temperature of 64° C., to anisolated nucleic acid molecule selected from the group consisting of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:16, or SEQ ID NO:18, whereinsaid oligonucleotide comprises at least about 50 nucleotides.

In another embodiment, a parasitic helminth cuticlin nucleic acidmolecule of the present invention includes a nucleic acid molecule thatis at least about 70%, more preferably at least about 75%, morepreferably at least about 80%, more preferably at least about 85%, morepreferably at least about 90%, and even more preferably at least about95% identical to nucleic acid molecule nDiCut-1A, nDiCut-1B, ornBmCut-1A, or an allelic variant of any of these nucleic acid molecules.Also preferred is a parasitic helminth cuticlin nucleic acid moleculecomprising a nucleic acid sequence that is that is at least about 70%,more preferably at least about 75%, more preferably at least about 80%,more preferably at least about 85%, more preferably at least about 90%,and even more preferably at least about 95% identical to nucleic acidsequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, or SEQ IDNO:18; or an allelic variant of a nucleic acid molecule having any ofthese sequences.

Particularly preferred is a cuticlin nucleic acid molecule comprisingall or part of nucleic acid molecule nDiCut-1A, nDiCut-1B, or nBmCut-1A,or an allelic variant of any these nucleic acid molecules. Alsoparticularly preferred is a nucleic acid molecule that includes at leasta portion of nucleic acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:16, or SEQ ID NO:18, or an allelic variant of a nucleic acidmolecule having any of these nucleic acid sequences. Such a nucleic acidmolecule can include nucleotides in addition to those included in theSEQ ID NOs, such as, but not limited to, nucleotides comprising afull-length gene, or nucleotides comprising a nucleic acid moleculeencoding a fusion protein or a nucleic acid molecule encoding amultivalent protective compound.

The present invention also includes a nucleic acid molecule encoding aprotein having at least a portion of SEQ ID NO:4, or an allelic variantof a nucleic acid molecule encoding a protein having at least a portionof SEQ ID NO:4. The present invention further includes a nucleic acidmolecule that has been modified to accommodate codon usage properties ofa cell in which such a nucleic acid molecule is to be expressed. Alsoincluded in the present invention is a nucleic acid molecule encoding aprotein having at least a portion of SEQ ID NO:9, or an allelic variantof a nucleic acid molecule encoding a protein having at least a portionof SEQ ID NO:9. The present invention further includes a nucleic acidmolecule that has been modified to accommodate codon usage properties ofa cell in which such a nucleic acid molecule is to be expressed. Alsoincluded in the present invention is a nucleic acid molecule encoding aprotein having at least a portion of SEQ ID NO:17, or an allelic variantof a nucleic acid molecule encoding a protein having at least a portionof SEQ ID NO:17. The present invention further includes a nucleic acidmolecule that has been modified to accommodate codon usage properties ofa cell in which such a nucleic acid molecule is to be expressed.

In another embodiment, a preferred parasitic helminth cuticlin nucleicacid molecule of the present invention comprises a nucleic acid moleculecomprising at least about 15 nucleotides, more preferably at least about18 nucleotides, more preferably at least about 20 nucleotides, morepreferably at least about 25 nucleotides, more preferably at least about30 nucleotides, more preferably at least about 40 nucleotides, morepreferably at least about 50 nucleotides, more preferably at least about100 nucleotides, more preferably at least about 150 nucleotides, morepreferably at least about 350 nucleotides, more preferably at leastabout 450 nucleotides, more preferably at least about 550 nucleotides,more preferably at least about 650 nucleotides, more preferably at leastabout 750 nucleotides, more preferably at least about 1000 nucleotides,more preferably at least about 1500 nucleotides, more preferably atleast about 1750 nucleotides more preferably at least about 1775nucleotides, and even more preferably at least about 2000 nucleotides inlength.

In another embodiment, a preferred parasitic helminth cuticlin nucleicacid molecule encodes a protein comprising at least about 5 amino acids,preferably at least about 6 amino acids, more preferably at least about10 amino acids, more preferably at least about 15 amino acids, morepreferably at least about 20 amino acids, more preferably at least about25 amino acids, more preferably at least about 30 amino acids, morepreferably at least about 40 amino acids, more preferably at least about50 amino acids, more preferably at least about 100 amino acids, morepreferably at least about 150 amino acids, more preferably at leastabout 200 amino acids, more preferably at least about 300 amino acids,more preferably at least about 375 amino acids, and even more preferablyat least about 400 amino acids in length.

Knowing the nucleic acid sequences of certain parasitic helminthcuticlin nucleic acid molecules of the present invention allows oneskilled in the art to, for example, (a) make copies of those nucleicacid molecules, (b) obtain nucleic acid molecules including at least aportion of such nucleic acid molecules (e.g., nucleic acid moleculesincluding full-length genes, full-length coding regions, regulatorycontrol sequences, truncated coding regions), and (c) obtain otherparasitic helminth cuticlin nucleic acid molecules. Such nucleic acidmolecules can be obtained in a variety of ways including screeningappropriate expression libraries with antibodies of the presentinvention; traditional cloning techniques using oligonucleotide probesof the present invention to screen appropriate libraries; and PCRamplification of appropriate libraries or DNA using oligonucleotideprimers of the present invention. Preferred libraries to screen or fromwhich to amplify nucleic acid molecules include Dirofilaria or B. malayiL3, L4 or adult cDNA libraries as well as genomic DNA libraries.Similarly, preferred DNA sources from which to amplify nucleic acidmolecules include Dirofilaria or B. malayi L3, L4 or adult first-strandcDNA syntheses and genomic DNA. Techniques to clone and amplify genesare disclosed, for example, in Sambrook et al., ibid.

The present invention also includes a nucleic acid molecule that is anoligonucleotide capable of hybridizing, under stringent hybridizationconditions, with complementary regions of other, preferably longer,nucleic acid molecules of the present invention such as those comprisingparasitic helminth cuticlin nucleic acid molecules; or withcomplementary regions of other parasitic helminth cuticlin nucleic acidmolecules. An oligonucleotide of the present invention can be RNA, DNA,or derivatives of either. The minimum size of such an oligonucleotide isthe size required for formation of a stable hybrid between theoligonucleotide and a complementary sequence on another nucleic acidmolecule. A preferred oligonucleotide of the present invention has amaximum size of about 100 nucleotides. The present invention includesoligonucleotides that can be used as, for example, probes to identifynucleic acid molecules, primers to produce nucleic acid molecules, ortherapeutic reagents to inhibit parasitic helminth cuticlin proteinproduction or activity (e.g., as antisense-, triplex formation-,ribozyme- and/or RNA drug-based reagents). The present invention alsoincludes the use of such oligonucleotides to protect animals fromdisease using one or more of such technologies. Appropriateoligonucleotide-containing therapeutic compositions can be administeredto an animal using techniques known to those skilled in the art.

Another embodiment of the present invention includes a recombinantvector, which includes at least one isolated nucleic acid molecule ofthe present invention inserted into any vector capable of delivering thenucleic acid molecule into a host cell. Such a vector containsheterologous nucleic acid sequences, that is, nucleic acid sequencesthat are not naturally found adjacent to nucleic acid molecules of thepresent invention, and that preferably are derived from a species otherthan the species from which the nucleic acid molecule(s) are derived.The vector can be either RNA or DNA, either prokaryotic or eukaryotic,and typically is a virus or a plasmid. Recombinant vectors can be usedto clone, sequence, or otherwise manipulate a parasitic helminthcuticlin nucleic acid molecule of the present invention.

One type of recombinant vector, referred to herein as a recombinantmolecule, comprises a nucleic acid molecule of the present inventionoperatively linked to an expression vector. The phrase “operativelylinked” refers to insertion of a nucleic acid molecule into anexpression vector in a manner such that the molecule is able to beexpressed when transformed into a host cell. As used herein, anexpression vector is a DNA or RNA vector that is capable of transforminga host cell and of effecting expression of a specified nucleic acidmolecule. Preferably, the expression vector is also capable ofreplicating within the host cell. An expression vector can be eitherprokaryotic or eukaryotic, and is typically a virus or a plasmid. Anexpression vector of the present invention includes any vector thatfunctions (i.e., directs gene expression) in a recombinant cell of thepresent invention, including in a bacterial, fungal, parasite, insect,other animal, or plant cell. A preferred expression vector of thepresent invention can direct gene expression in a bacterial, yeast,helminth or other parasite, insect or mammalian cell, or more preferablyin a cell type disclosed herein.

In particular, an expression vector of the present invention containsregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell andthat control the expression of a nucleic acid molecule of the presentinvention. In particular, a recombinant molecule of the presentinvention includes transcription control sequences. Transcriptioncontrol sequences are sequences which control the initiation,elongation, and termination of transcription. Particularly importanttranscription control sequences are those which control transcriptioninitiation, such as promoter, enhancer, operator and repressorsequences. A suitable transcription control sequence includes anytranscription control sequence that can function in at least one of therecombinant cells of the present invention. A variety of suchtranscription control sequences are known to those skilled in the art.Preferred transcription control sequences include those which functionin bacterial, yeast, helminth or other parasite, insect or mammaliancells, such as, but not limited to, tac, lac, trp, trc, oxy-pro,omp/lpp, rrnB, bacteriophage lambda (such as lambda p_(L) and lambdap_(R) and fusions that include such promoters), bacteriophage T7, T7lac,bacteriophage T3, bacteriophage SP6, bacteriophage SP01,metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirussubgenomic promoters (such as Sindbis virus subgenomic promoters),antibiotic resistance gene, baculovirus, Heliothis zea insect virus,vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus,adenovirus, cytomegalovirus (such as immediate early promoters),picornavirus, simian virus 40, retrovirus, actin, retroviral longterminal repeat, Rous sarcoma virus, heat shock, phosphate or nitratetranscription control sequences; as well as other sequences capable ofcontrolling gene expression in prokaryotic or eukaryotic cells.Additional suitable transcription control sequences includetissue-specific promoters and enhancers as well as lymphokine-induciblepromoters (e.g., promoters inducible by interferons or interleukins).Transcription control sequences of the present invention can alsoinclude naturally occurring transcription control sequences naturallyassociated with parasitic helmintis, such as D. immitis or B. malayi.

Suitable and preferred nucleic acid molecules to include in arecombinant vector of the present invention are as disclosed herein.Preferred nucleic acid molecules to include in a recombinant vector, andparticularly in a recombinant molecule, include nDiCut-1A, nDiCut-1B, ornBmCut-1A, the production of which are described in the Examplessection.

A recombinant molecule of the present invention may also (a) contain asecretory signal (i.e., a signal segment nucleic acid sequence) toenable an expressed cuticlin protein of the present invention to besecreted from the cell that produces the protein or (b) contain a fusionsequence which leads to the expression of a nucleic acid molecule of thepresent invention as a fusion protein. Examples of suitable signalsegments include any signal segment capable of directing the secretionof a protein of the present invention. Preferred signal segmentsinclude, but are not limited to, native parasitic helminth signalsegments, tissue plasminogen activator (t-PA), interferon, interleukin,growth hormone, histocompatibility and viral envelope glycoproteinsignal segments. Suitable fusion segments encoded by fusion segmentnucleic acids are disclosed herein. In addition, a nucleic acid moleculeof the present invention can be joined to a fusion segment that directsthe encoded protein to the proteosome, such as a ubiquitin fusionsegment. A eukaryotic recombinant molecule may also include interveningand/or untranslated sequences surrounding and/or within the nucleic acidsequence of the nucleic acid molecule of the present invention.

Another embodiment of the present invention includes a recombinant cellcomprising a host cell transformed with one or more recombinantmolecules of the present invention. Transformation of a nucleic acidmolecule into a cell can be accomplished by any method by which anucleic acid molecule can be inserted into the cell. Transformationtechniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. A recombinant cell may remain unicellular or may grow into atissue, organ, or a multicellular organism. Transformed nucleic acidmolecules of the present invention can remain extrachromosomal or canintegrate into one or more sites within a chromosome of the transformed(i.e., recombinant) cell in such a manner that their ability to beexpressed is retained. Preferred nucleic acid molecules with which totransform a cell include cuticlin nucleic acid molecules disclosedherein. Particularly preferred nucleic acid molecules with which totransform a cell include nDiCut-1A, nDiCut-1B, and nBmCut-1A.

Suitable host cells to transform include any cell that can betransformed with a nucleic acid molecule of the present invention. Hostcells can be either untransformed cells or cells that are alreadytransformed with at least one nucleic acid molecule (e.g., nucleic acidmolecules encoding one or more proteins of the present invention orencoding other proteins useful in the production of multivalentvaccines). A recombinant cell of the present invention can beendogenously (i.e., naturally) capable of producing a parasitic helminthcuticlin protein of the present invention or can be capable of producingsuch a protein after being transformed with at least one nucleic acidmolecule of the present invention. A host cell of the present inventioncan be any cell capable of producing at least one protein of the presentinvention, and can be a bacterial, fungal (including yeast), parasite(including helminth, protozoa and ectoparasite), other insect, otheranimal or plant cell. Preferred host cells include bacterial,mycobacterial, yeast, helminth, insect and mammalian cells. Morepreferred host cells include Salmonella, Escherichia, Bacillus,Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK(baby hamster kidney) cells, MDCK cells (Madin-Darby Canine Kidneycells), CRFK cells (Crandell Feline Kidney cells), BSC-1 cells (Africanmonkey kidney cell line used, for example, to culture poxviruses), COS(e.g., COS-7) cells, and Vero cells. Particularly preferred host cellsare Escherichia coli, including E. colit K-12 derivatives; Salmonellatyphi; Salmonella typhimurium, including attenuated strains such asUK-1_(χ)3987 and SR-11_(χ)4072; Spodoptera frugiperda; Trichoplusia ni;BHK cells; MDCK cells; CRFK cells; BSC-1 cells; COS cells; Vero cells;and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246).Additional appropriate mammalian cell hosts include other kidney celllines, other fibroblast cell lines (e.g., human, murine or chickenembryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovarycells, mouse NIH/3T3 cells, LMTK³¹ cells and/or HeLa cells. In oneembodiment, the proteins may be expressed as heterologous proteins inmyeloma cell lines employing immunoglobulin promoters.

A recombinant cell of the present invention includes any celltransformed with at least one of any nucleic acid molecule of thepresent invention. Suitable and preferred nucleic acid molecules as wellas suitable and preferred recombinant molecules with which to transformsuch a cell are disclosed herein.

In one embodiment, a recombinant cell of the present invention can beco-transformed with a recombinant molecule including a parasitichelminth cuticlin nucleic acid molecule encoding a protein of thepresent invention and a nucleic acid molecule encoding anotherprotective compound, as disclosed herein (e.g., to produce multivalentvaccines).

Recombinant DNA technologies can be used to improve expression of atransformed nucleic acid molecule by manipulating, for example, thenumber of copies of the nucleic acid molecule within a host cell, theefficiency with which that nucleic acid molecule is transcribed, theefficiency with which the resultant transcript is translated, and theefficiency of post-translational modifications. Recombinant techniquesuseful for increasing the expression of a nucleic acid molecule of thepresent invention include, but are not limited to, operatively linkingthe nucleic acid molecule to a high-copy number plasmid, integration ofthe nucleic acid molecule into one or more host cell chromosomes,addition of vector stability sequences to a plasmid, substitution ormodification of transcription control signals (e.g., promoters,operators, enhancers), substitution or modification of translationalcontrol signals (e.g., ribosome binding sites, Shine-Dalgarno sequences,or Kozak sequences), modification of a nucleic acid molecule of thepresent invention to correspond to the codon usage of the host cell,deletion of sequences that destabilize transcripts, and the use ofcontrol signals that temporally separate recombinant cell growth fromrecombinant enzyme production during fermentation. The activity of anexpressed recombinant protein of the present invention may be improvedby fragmenting, modifying, or derivatizing a nucleic acid moleculeencoding such a protein.

Isolated parasitic helminth cuticlin proteins of the present inventioncan be produced in a variety of ways, including production and recoveryof natural proteins, production and recovery of recombinant proteins,and chemical synthesis of the proteins. In one embodiment, an isolatedprotein of the present invention is produced by culturing a cell capableof expressing the protein under conditions effective to produce theprotein, and recovering the protein. A preferred cell to culture is arecombinant cell of the present invention. Effective culture conditionsinclude, but are not limited to, effective media, bioreactor,temperature, pH and oxygen conditions that permit protein production. Aneffective medium refers to any medium in which a cell is cultured toproduce a parasitic helminth cuticlin protein of the present invention.Such a medium typically comprises an aqueous base having assimilablecarbon, nitrogen and phosphate sources, and appropriate salts, minerals,metals and other nutrients, such as vitamins. Cells of the presentinvention can be cultured in conventional fermentation bioreactors,shake flasks, test tubes, microtiter dishes, and petri plates. Culturingcan be carried out at a temperature, pH and oxygen content appropriatefor a given recombinant cell. Such culturing conditions are within theexpertise of one of ordinary skill in the art. Examples of suitableconditions are included in the Examples section.

Depending on the vector and host system used for production, a resultantprotein of the present invention may either remain within therecombinant cell; be secreted into the fermentation medium; be secretedinto a space between two cellular membranes, such as the periplasmicspace in E. coli; or be retained on the outer surface of a cell or viralmembrane.

The phrase “recovering the protein”, as well as similar phrases, referto collecting the whole fermentation medium containing the protein andneed not imply additional steps of separation or purification. Proteinsof the present invention can be purified using a variety of standardprotein purification techniques, such as, but not limited to, affinitychromatography, ion exchange chromatography, filtration,electrophoresis, hydrophobic interaction chromatography, gel filtrationchromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.Proteins of the present invention are preferably retrieved in“substantially pure” form. As used herein, “substantially pure” refersto a purity that allows for the effective use of the protein as atherapeutic composition or diagnostic. A therapeutic composition foranimals, for example, should exhibit no substantial toxicity andpreferably should be capable of stimulating the production of antibodiesin a treated animal.

The present invention also includes isolated (i.e., removed from theirnatural milieu) antibodies that selectively bind to a parasitic helminthcuticlin protein of the present invention or a mimetope thereof (e.g.,anti-parasitic helminth cuticlin antibodies). As used herein, the term“selectively binds to” a cuticlin protein refers to the ability of anantibody of the present invention to preferentially bind to specifiedproteins and mimetopes thereof of the present invention. Binding can bemeasured using a variety of methods standard in the art including enzymeimmunoassays (e.g., ELISA), immunoblot assays, etc. See, for example,Sambrook et al., ibid., and Harlow, et al., 1988, Antibodies, aLaboratory Manual, Cold Spring Harbor Labs Press; Harlow et al., ibid.,is incorporated by reference herein in its entirety. An anti-parasitichelminth cuticlin antibody preferably selectively binds to a parasitichelminth cuticlin protein in such a way as to reduce the activity ofthat protein.

Isolated antibodies of the present invention can include antibodies inserum, or antibodies that have been purified to varying degrees.Antibodies of the present invention can be polyclonal or monoclonal,functional equivalents such as antibody fragments andgenetically-engineered antibodies, including single chain antibodies orchimeric antibodies that can bind to more than one epitope.

A preferred method to produce antibodies of the present inventionincludes (a) administering to an animal an effective amount of aprotein, peptide or mimetope thereof of the present invention to producethe antibodies and (b) recovering the antibodies. In another method,antibodies of the present invention are produced recombinantly usingtechniques as heretofore disclosed to produce cuticlin proteins of thepresent invention. Antibodies raised against defined proteins ormimetopes can be advantageous because such antibodies are notsubstantially contaminated with antibodies against other substances thatmight otherwise cause interference in a diagnostic assay or side effectsif used in a therapeutic composition.

Antibodies of the present invention have a variety of potential usesthat are within the scope of the present invention. For example, suchantibodies can be used (a) as therapeutic compounds to passivelyimmunize an animal in order to protect the animal from parasitichelminths susceptible to treatment by such antibodies, (b) as reagentsin assays to detect infection by such helminths or (c) as tools toscreen expression libraries or to recover desired proteins of thepresent invention from a mixture of proteins and other contaminants.Furthermore, antibodies of the present invention can be used to targetcytotoxic agents to parasitic helminths of the present invention inorder to directly kill such helminths. Targeting can be accomplished byconjugating (i.e., stably joining) such antibodies to the cytotoxicagents using techniques known to those skilled in the art. Suitablecytotoxic agents are known to those skilled in the art.

One embodiment of the present invention is a therapeutic compositionthat, when administered to an animal in an effective manner, is capableof protecting that animal from disease caused by a parasitic helminth. Atherapeutic composition of the present invention includes an excipientand at least one of the following protective compounds: an isolatednative parasitic helminth cuticlin protein; an isolated non-nativeparasitic helminth cuticlin protein; a mimetope of a parasitic helminthcuticlin protein; an isolated parasitic helminth cuticlin nucleic acidmolecule; an isolated antibody that selectively binds to a parasitichelminth cuticlin protein; or an inhibitor of cuticlin protein activityidentified by its ability to inhibit parasitic helminth cuticlinactivity. As used herein, a protective compound refers to a compoundthat, when administered to an animal in an effective manner, is able totreat, ameliorate, or prevent disease caused by a parasitic helminth.Preferred helminths to target are heretofore disclosed. Examples ofproteins, nucleic acid molecules, antibodies and inhibitors of thepresent invention are disclosed herein.

The present invention also includes a therapeutic composition comprisingat least one parasitic helminth cuticlin-based compound of the presentinvention in combination with at least one additional compoundprotective against one or more infectious agents. Examples of suchcompounds and infectious agents are disclosed herein.

A therapeutic composition of the present invention can be administeredto any animal susceptible to such therapy, preferably to mammals, andmore preferably to dogs, cats, humans, ferrets, horses, cattle, sheepand other pets, work animals, economic food animals, or zoo animals.Preferred animals to protect against heartworm disease include dogs,cats, humans and ferrets, with dogs and cats being particularlypreferred.

In one embodiment, a therapeutic composition of the present inventioncan be administered to the vector in which the parasitic helminthdevelops, such as to a mosquito, in order to prevent the spread ofparasitic helminth to the definitive mammalian host. Such administrationcould be orally or by developing transgenic vectors capable of producingat least one therapeutic composition of the present invention. Inanother embodiment, a vector, such as a mosquito, can ingest therapeuticcompositions present in the blood of a host that has been administered atherapeutic composition of the present invention.

A therapeutic composition of the present invention can be formulated inan excipient that the animal to be treated can tolerate. Examples ofsuch excipients include water, saline, Ringer's solution, dextrosesolution, Hank's solution, and other aqueous physiologically balancedsalt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil,ethyl oleate, or triglycerides may also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer, and Tris buffer, while examples ofpreservatives include thimerosal,—or o-cresol, formalin, and benzylalcohol. Standard formulations can either be liquid injectables orsolids which can be taken up in a suitable liquid as a suspension orsolution for injection. Thus, in a non-liquid formulation, the excipientcan comprise dextrose, human serum albumin, preservatives, etc., towhich sterile water or saline can be added prior to administration.

In one embodiment of the present invention, a therapeutic compositioncan include an adjuvant. Adjuvants are agents that are capable ofenhancing the immune response of an animal to a specific antigen.Suitable adjuvants include, but are not limited to, cytokines,chemokines, and compounds that induce the production of cytokines andchemokines (e.g., granulocyte macrophage colony stimulating factor(GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophagecolony stimulating factor (M-CSF), colony stimulating factor (CSF),erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (IL-3),interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6),interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10),interleukin 12 (IL-12), interferon gamma, interferon gamma inducingfactor I (IGIF), transforming growth factor beta, RANTES (regulated uponactivation, normal T-cell expressed and presumably secreted), macrophageinflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), and Leishmaniaelongation initiating factor (LEIF)); bacterial components (e.g.,endotoxins, in particular superantigens, exotoxins and cell wallcomponents); aluminum-based salts; calcium-based salts; silica;polynucleotides; toxoids; serum proteins, viral coat proteins; blockcopolymer adjuvants (e.g., Hunter's Titermax™ adjuvant (Vaxcel™, Inc.Norcross, Ga.), Ribi adjuvants (Ribi ImmunoChem Research, Inc.,Hamilton, Mont.); and saponins and their derivatives (e.g., Quil A(Superfos Biosector A/S, Denmark). Protein adjuvants of the presentinvention can be delivered in the form of the protein themselves or ofnucleic acid molecules encoding such proteins using the methodsdescribed herein.

In one embodiment of the present invention, a therapeutic compositioncan include a carrier. Carriers include compounds that increase thehalf-life of a therapeutic composition in the treated animal. Suitablecarriers include, but are not limited to, polymeric controlled releasevehicles, biodegradable implants, liposomes, bacteria, viruses, othercells, oils, esters, and glycols.

One embodiment of the present invention is a controlled releaseformulation that is capable of slowly releasing a composition of thepresent invention into an animal. As used herein, a controlled releaseformulation comprises a composition of the present invention in acontrolled release vehicle. Suitable controlled release vehiclesinclude, but are not limited to, biocompatible polymers, other polymericmatrices, capsules, microcapsules, microparticles, bolus preparations,osmotic pumps, diffusion devices, liposomes, lipospheres, andtransdermal delivery systems. Other controlled release formulations ofthe present invention include liquids that, upon administration to ananimal, form a solid or a gel in situ, Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation of the present invention iscapable of releasing a composition of the present invention into theblood of the treated animal at a constant rate sufficient to attaintherapeutic dose levels of the composition to protect an animal fromdisease caused by parasitic helminths. The therapeutic composition ispreferably released over a period of time ranging from about 1 to about12 months. A controlled release formulation of the present invention iscapable of effecting a treatment preferably for at least about 1 month,more preferably for at least about 3 months, even more preferably for atleast about 6 months, even more preferably for at least about 9 months,and even more preferably for at least about 12 months.

In order to protect an animal from disease caused by a parasitichelminth, a therapeutic composition of the present invention isadministered to the animal in an effective manner such that thecomposition is capable of protecting that animal from a disease causedby a parasitic helminth. For example, an isolated protein or mimetopethereof is administered in an amount and manner that elicits (i.e.,stimulates) an immune response that is sufficient to protect the animalfrom the disease. Similarly, an antibody of the present invention, whenadministered to an animal in an effective manner, is administered in anamount so as to be present in the animal at a titer that is sufficientto protect the animal from the disease, at least temporarily. Anoligonucleotide nucleic acid molecule of the present invention can alsobe administered in an effective manner, thereby reducing expression ofnative parasitic helminth cuticlin proteins in order to interfere withdevelopment of the parasitic helminths targeted in accordance with thepresent invention.

Therapeutic compositions of the present invention can be administered toanimals prior to infection in order to prevent infection (i.e., as apreventative vaccine) or can be administered to animals after infectionin order to treat disease caused by the parasitic helminth (i.e., as acurative agent or a therapeutic vaccine).

Acceptable protocols to administer therapeutic compositions in aneffective manner include individual dose size, number of doses,frequency of dose administration, and mode of administration.Determination of such protocols can be accomplished by those skilled inthe art. A suitable single dose is a dose that is capable of protectingan animal from disease when administered one or more times over asuitable time period. For example, a preferred single dose of a protein,mimetope, or antibody therapeutic composition is from about 1 microgram(μg) to about 10 milligrams (mg) of the therapeutic composition perkilogram body weight of the animal. Booster vaccinations can beadministered from about 2 weeks to several years after the originaladministration. Booster administrations preferably are administered whenthe immune response of the animal becomes insufficient to protect theanimal from disease. A preferred administration schedule is one in whichfrom about 10 μg to about 1 mg of the therapeutic composition per kgbody weight of the animal is administered from about one to about twotimes over a time period of from about 2 weeks to about 12 months. Modesof administration can include, but are not limited to, subcutaneous,intradermal, intravenous, intranasal, oral, transdermal, andintramuscular routes.

According to one embodiment, a nucleic acid molecule of the presentinvention can be administered to an animal in a fashion to enableexpression of that nucleic acid molecule into a protective protein orprotective RNA (e.g., an antisense RNA, a ribozyme, a triple helix form,or an RNA drug) in the animal. Nucleic acid molecules can be deliveredto an animal by a variety of methods including, but not limited to, (a)administering a genetic vaccine (e.g., a naked DNA or RNA molecule, suchas is taught, for example, in Wolff et al., 1990, Science 247,1465-1468) or (b) administering a nucleic acid molecule packaged as arecombinant virus vaccine or as a recombinant cell vaccine (i.e., thenucleic acid molecule is delivered by a viral or cellular vehicle).

A genetic (i.e., naked nucleic acid) vaccine of the present inventionincludes a nucleic acid molecule of the present invention and preferablyincludes a recombinant molecule of the present invention that preferablyis replication, or otherwise amplification, competent. A genetic vaccineof the present invention can comprise one or more nucleic acid moleculesof the present invention in the form of, for example, a dicistronicrecombinant molecule. A preferred genetic vaccine includes at least aportion of a viral genome (i.e., a viral vector). Preferred viralvectors include those based on alphaviruses, poxviruses, adenoviruses,herpesviruses, picornaviruses, and retroviruses, with those based onalphaviruses (such as Sindbis or Semliki forest virus), species-specificherpesviruses and poxviruses being particularly preferred. Any suitabletranscription control sequence can be used, including those disclosed assuitable for protein production. Particularly preferred transcriptioncontrol sequences include cytomegalovirus immediate early (preferably inconjunction with Intron-A), Rous sarcoma virus long terminal repeat, andtissue-specific transcription control sequences, as well astranscription control sequences endogenous to viral vectors if viralvectors are used. The incorporation of “strong” poly(A) sequences isalso preferred.

A genetic vaccine of the present invention can be administered in avariety of ways, with intramuscular, subcutaneous, intradermal,transdermal, intranasal and oral routes of administration beingpreferred. A preferred single dose of a genetic vaccine ranges fromabout 1 nanogram (ng) to about 500 μg, depending on the route ofadministration or method of delivery, as can be determined by thoseskilled in the art. Suitable delivery methods include, for example, byinjection, as drops, aerosolized, or topically. Genetic vaccines of thepresent invention can be contained in an aqueous excipient (e.g.,phosphate buffered saline) alone or in a carrier (e.g., lipid-basedvehicles).

A recombinant virus vaccine of the present invention includes arecombinant molecule of the present invention that is packaged in aviral coat and that can be expressed in an animal after administration.Preferably, the recombinant molecule is packaging- orreplication-deficient or encodes an attenuated virus. A number ofrecombinant viruses can be used, including, but not limited to, thosebased on alphaviruses, poxviruses, adenoviruses, herpesviruses,picornaviruses, and retroviruses. Preferred recombinant virus vaccinesare those based on alphaviruses (such as Sindbis virus), raccoonpoxviruses, picornaviruses, and species-specific herpesviruses. Methodsto produce and use a recombinant alphavirus vaccine are disclosed in PCTPublication No. WO 94/17813, by Xiong et al., published Aug. 18, 1994,which is incorporated by reference herein in its entirety.

When administered to an animal, a recombinant virus vaccine of thepresent invention infects cells within the immunized animal and directsthe production of a protective protein or RNA nucleic acid molecule thatis capable of protecting the animal from disease caused by a parasitichelminth as disclosed herein. For example, a recombinant virus vaccinecomprising a parasitic helminth cuticlin nucleic acid molecule of thepresent invention is administered according to a protocol that resultsin the animal producing a sufficient immune response to protect itselffrom heartworm disease. A preferred single dose of a recombinant virusvaccine of the present invention is from about 1×10⁴ to about 1×10⁸virus plaque forming units (pfu) per kilogram body weight of the animal.Administration protocols are similar to those described herein forprotein-based vaccines, with subcutaneous, intramuscular, intranasal andoral administration routes being preferred.

A recombinant cell vaccine of the present invention includes arecombinant cell of the present invention that expresses at least oneprotein of the present invention. Preferred recombinant cells for thisembodiment include Salmonella, E. coli, Listeria, Mycobacterium, S.frugiperda, yeast (including Saccharomyces cerevisiae and Pichiapastoris), BHK, BSC-1, myoblast G8, COS (e.g., COS-7), Vero, MDCK orCRFK recombinant cells. A recombinant cell vaccine of the presentinvention can be administered in a variety of ways but has the advantagethat it can be administered orally, preferably at doses ranging fromabout 10⁸ to about 10¹² cells per kilogram body weight. Administrationprotocols are similar to those described herein for protein-basedvaccines. A recombinant cell vaccine can comprise whole cells, cellsstripped of cell walls or cell lysates.

The efficacy of a therapeutic composition of the present invention toprotect an animal from disease caused by a parasitic helminth can betested in a variety of ways including, but not limited to, detection ofprotective antibodies (using, for example, proteins or mimetopes of thepresent invention), detection of cellular immunity within the treatedanimal, or challenge of the treated animal with the parasitic helminthto determine whether the treated animal is resistant to disease.Challenge studies can include implantation of chambers includingparasitic helminth larvae into the treated animal and/or directadministration of larvae to the treated animal. In one embodiment,therapeutic compositions can be tested in animal models such as mice.Such techniques are known to those skilled in the art.

One preferred embodiment of the present invention is the use ofparasitic helminth cuticlin proteins, nucleic acid molecules, antibodiesor inhibitory compounds of the present invention to protect an animalfrom heartworm disease. It is particularly preferred to prevent L3 thatare delivered to the animal by the mosquito intermediate host frommaturing into adult worms. Accordingly, a preferred therapeuticcomposition is one that is able to inhibit at least one step in theportion of the parasite's development cycle that includes L3, thirdmolt, L4, fourth molt, and immature adult prior to entering thecirculatory system. In dogs, this portion of the developmental cycle isabout 70 days in length. A particularly preferred therapeuticcomposition includes a parasitic helminth cuticlin-based therapeuticcomposition of the present invention, particularly in light of theevidence herein reported that cuticlin is expressed in both larval andadult stages of the parasite. Such a composition can include a parasitichelminth cuticlin nucleic acid molecule, a parasitic helminth cuticlinprotein or a mimetope thereof, anti-parasitic helminth cuticlinantibodies, or inhibitors of parasitic helminth cuticlin activity. Suchtherapeutic compositions are administered to an animal in a mannereffective to protect the animals from heartworm disease. Additionalprotection may be obtained by administering additional protectivecompounds, including other parasitic helminth proteins, nucleic acidmolecules, antibodies and inhibitory compounds, as disclosed herein.

One therapeutic composition of the present invention includes aninhibitor of parasitic helminth cuticlin activity, i.e., a compoundcapable of substantially interfering with the function of a parasitichelminth cuticlin protein, also referred to herein as a cuticlininhibitor. In one embodiment, such an inhibitor comprises a compoundthat interacts directly with a cuticlin protein active site (usually bybinding to or modifying the active site), thereby inhibiting cuticlinactivity. According to this embodiment, a cuticlin inhibitor can alsointeract with other regions of a cuticlin protein to inhibit cuticlinactivity, for example, by allosteric interaction. Preferably, a cuticlininhibitor of the present invention is identified by its ability to bindto, or otherwise interact with, a parasitic helminth cuticlin protein,thereby inhibiting cuticlin activity of that protein. Such a cuticlininhibitor is a suitable for inclusion in a therapeutic composition ofthe present invention as long as the compound is not harmful to the hostanimal being treated.

A cuticlin inhibitor can be identified using a parasitic helminthcuticlin protein of the present invention. As such, one embodiment ofthe present invention is a method to identify a compound capable ofinhibiting cuticlin activity of a parasitic helminth susceptible toinhibition by an inhibitor of parasitic helminth cuticlin activity. Sucha method includes the steps of (a) contacting (e.g., combining, mixing)an isolated parasitic helminth cuticlin protein, preferably a D. immitiscuticlin protein, with a putative inhibitory compound under conditionsin which, in the absence of the compound, the protein has cuticlinactivity, and (b) determining if the putative inhibitory compoundinhibits the cuticlin activity. Putative inhibitory compounds to screeninclude small organic molecules, antibodies (including mimetopesthereof) and substrate analogs. Methods to determine cuticlin activityare known to those skilled in the art; see, for example, Rhee, et al.,ibid., Lim, et al., ibid., Sauri, et al., ibid., and Kim, et al., ibid.

The present invention also includes a test kit to identify a compoundcapable of inhibiting cuticlin activity of a parasitic helminth. Such atest kit includes an isolated parasitic helminth cuticlin protein,preferably a D. immitis cuticlin protein, having cuticlin activity, anda means for determining the extent of inhibition of cuticlin activity inthe presence of (i.e., effected by) a putative inhibitory compound. Suchcompounds are also screened to identify those that are substantially nottoxic in host animals, e.g., compounds that do not inhibit the activityof mammalian cuticlin.

Cuticlin inhibitors isolated by such a method or test kit can be used toinhibit any parasitic helminth cuticlin protein that is susceptible tosuch an inhibitor. A particularly preferred cuticlin inhibitor of thepresent invention is capable of protecting an animal from heartwormdisease. A therapeutic composition comprising a compound that inhibitscuticlin activity can be administered to an animal in an effectivemanner to protect that animal from disease caused by the parasiteexpressing the targeted cuticlin enzyme, and preferably to protect thatanimal from heartworm disease. Effective amounts and dosing regimens canbe determined using techniques known to those skilled in the art.

It is also within the scope of the present invention to use isolatedproteins, mimetopes, nucleic acid molecules and antibodies of thepresent invention as diagnostic reagents to detect infection byparasitic helminths. Such diagnostic reagents can be supplemented withadditional compounds that can detect specific phases of the parasite'slife cycle. Methods to use such diagnostic reagents to diagnoseparasitic helminth infection are well known to those skilled in the art.Suitable and preferred parasitic helminths to detect are those to whichtherapeutic compositions of the present invention are targeted.Particularly preferred parasitic helminths to detect using diagnosticreagents of the present invention are D. immitis or B. malayi.

A Sequence Listing pursuant to 37 CFR §1.821 is submitted herewith onseparately numbered pages. A copy in computer readable form is alsosubmitted herewith. Applicants assert pursuant to 37 CFR §1.821(f) thatthe content of the paper and computer readable copies of SEQ ID NO:1through SEQ ID NO:18 submitted herewith are the same.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLES

It is to be noted that these Examples include a number of molecularbiology, microbiology, immunology and biochemistry techniques familiarto those skilled in the art. Disclosure of such techniques can be found,for example, in Sambrook et al., ibid., Ausubel, et al., 1993, CurrentProtocols in Molecular Biology, (Greene/Wiley Interscience, New York,N.Y., and related references. Ausubel, et al, ibid. is incorporated byreference herein in its entirety. DNA and protein sequence analyses werecarried out using the PC/GENE™ sequence analysis program, version 6.60(available from Intelligenetics, Inc., Mountainview, Calif.). Thesettings for analysis were as follows. NAlign:(nucleic acid) open gapcost 10; unit gap cost −10; Palign(protein): open gap cost −2, unit gapcost −2; CLUSTAL: Protein, K-triple for protein=1, Gap penalty=10,Window size=10; Nucleic acid, K-triple for nucleic acids=5; filteringlevel=2.5; Parameter for final alignment: Open gap cost=10, Unit gapcost=10; Transitions are WEIGHTED twice as likely as transversions. Itshould also be noted that, because nucleic acid sequencing technology,and in particular the sequencing of PCR products, is not entirelyerror-free, the nucleic acid and deduced protein sequences presentedherein represent apparent nucleic acid sequences of the nucleic acidmolecules encoding parasitic helminth cuticlin proteins of the presentinvention.

Example 1

This example describes the molecular cloning of two related cuticlingenes from Dirofilaria immitis, referred to herein as DiCut-1A andDiCut-1B. As is herein described, DiCut-1A and DiCut-1B are related tothe cut-1 cuticlin genes from Ascaris and C. elegans. Despite thisrelatedness, initial attempts to isolate genes encoding cuticlinproteins from D. immitis using degenerate primers based on C. elegansand Ascaris cut-1 were unsuccessful. Subsequent to the unsuccessfulattempts to isolate cuticlin genes from D. immitis based on homology tothe Ascaris and C. elegans cut-1 cuticlin genes, D. immitis cDNAsequences with homology to the Ascaris and C. elegans cut-1 genes wereidentified from a D. immitis larval EST DNA sequence, as follows.

D. immitis larval cDNA was produced and enriched for DNA representinglarval message in relation to adult message using a CLONTECH PCR-Select™cDNA subtraction kit (available from CLONETECH, Palo Alto, Calif.)according to the manufacturer's instructions. These larval cDNAs weresubcloned into the pCRII™ vector (available from Invitrogen, San Diego,Calif.) according to the manufacturer's instructions. The isolated andsubcloned larval cDNAs were sequenced by the Sanger dideoxy chaintermination method, using the PRISM™ Ready Dye Terminator CycleSequencing Kit with AmpliTaq® DNA Polymerase, FS (available from thePerkin-Elmer Corporation, Norwalk, Conn.). PCR extensions were done inthe GeneAmp™ PCR System 9600 (available from Perkin-Elmer). Excess dyeterminators were removed from extension products using the Centriflex™Gel Filtration Cartridge (available from Advanced Genetics TechnologiesCorporation, Gaithersburg, Md.) following the standard protocol. Sampleswere resuspended according to ABI protocols and were run on aPerkin-Elmer ABI PRISM™ 377 Automated DNA Sequencer.

The first EST sequence obtained represented a D. immitis cuticlinnucleic acid molecule 1016 bp long. This sequence comprises the nucleicacid sequence between base pairs 194 and 1210 of the essentiallyfull-length cDNA sequence of a D. immitis cuticlin nucleic acid moleculereferred to herein as nDiCut-1A (the coding and complementary strands ofwhich are herein represented by SEQ ID NO:1 and SEQ ID NO:2,respectively). A second D. immitis cuticlin EST fragment was obtainedwhich overlapped with the previous EST fragment at the 3′ end, andcontributed an additional 569 base pairs of sequence comprising thenucleotide sequence between base pairs 1211 and 1779 of nDiCut-1A (SEQID NO:1). In order to obtain the 5′ end of nDiCut-1A, and to confirm thesequence of the 3′ end of the molecule, sense and antisense primersspecific to the D. immitis cuticlin EST sequence were designed tohybridize to nDiCut-1A between base pairs 862 and 895 of the finalnDiCut-1A sequence (SEQ ID NO:1). The sense primer specific to the EST,referred to herein as CUT-3′R (SEQ ID NO:11), consists of the sequence:5′ G GCT GGC CAA GAA GCT CAC GTA TAC AAA TAT GCG 3′. The antisenseprimer referred to herein as CUT-5′R (SEQ ID NO:12), consists of thesequence: 5′ CGC ATA TTT GTA TAC GTG AGC TTC TTG GCC AGC C 3′. The 5′end of the cuticlin EST nucleic acid molecule was amplified by standardRT-PCR methods from D. immitis L3-48 hr first-strand cDNA using thenematode 22-bp splice leader sequence, referred to herein as SL1 (5′GGTTTAATTA CCCAAGTTTG AG 3′; SEQ ID NO:13) and the EST-specificantisense primer (SEQ ID NO:12). The RT-PCR reaction generated an 895 bpproduct. The composite full length cDNA sequence of nDiCut-1A comprisesa 1779 bp nucleic acid molecule (the coding and complementary strands ofwhich are herein represented by SEQ ID NO:1 and SEQ ID NO:2,respectively). nDiCut-1A encodes a 387 amino acid protein (hereinreferred to as PDiCut-1A, represented by SEQ ID NO:4).

RT-PCR using SL1 (SEQ ID NO:13) and the EST-specific antisense primer,CUT-5′R (SEQ ID NO:12) was also carried out using Brugia malayi adultfemale cDNA as a template. This reaction resulted in a partial 5′ endproduct of a Brugia cuticlin homolog, referred to herein as nBmcut-1A(the coding strand and reverse complement of which are hereinrepresented by SEQ ID NO:16 and SEQ ID NO: 8, respectively). nBmCut-1Aencodes a 245 amino acid protein (herein referred to as PBmCut-1A,represented by SEQ ID NO:17).

In order to confirm the sequence of the 3′ nDiCut-1A EST fragment, 3′RACE PCR was performed using a Marathon™ cDNA Amplification Kit(available from CLONTECH) according to the manufacturer's instructions.The template for amplification was D. immitis adult female first-strandcDNA and amplification was performed using the EST-specific senseprimer, CUT-3′R (SEQ ID NO:11) and an antisense RACE-adapter primer (5′CCA TCC TAA TAC GAC TCA CTA TAG GGC 3′, referred to herein as SEQ IDNO:14). Instead of obtaining a product of the predicted size of 919 bp(as would be expected if the amplified product represented nDiCut-1Asequence), a 643 bp nucleic acid molecule was obtained. This moleculerepresented the 3′ end of an additional D. immitis cuticlin nucleic acidmolecule referred to herein as nDiCut-1B. The 3′ sequence of nDiCut-1Bwas very different from the sequence already determined for the 39 endof nDiCut-1A. The 5′ end of nDiCut-1B was amplified by SL1 RT-PCR usingan nDiCut-1B specific antisense primer. This DiCut-1B primer, referredto as CUTb, consists of the sequence: GGT TAT ATC AAC COT GCT AAA ACCGGT ACT GAC GTC CAC CG (herein referred to as SEQ ID NO:15), andrepresents the nucleic acid sequence located between base pairs 981 and1020 of the essentially full-length nDiCut-1B cDNA sequence (SEQ IDNO:6). RT-PCR using CUTb and SL1 as primers generated a 1020 bpnDiCut-1B sequence when either larval or adult first-strand cDNA wasused as the template. The composite full length cDNA sequence ofntiCut-1B comprises 1372 bp, herein represented by SEQ ID NO:6 (thecoding strand) and SEQ ID NO:7 (the reverse complement). nDiCut-1Bencodes a271 amino acids (herein referred to as PDiCut-1B, representedby SEQ ID NO:9).

RT-PCR reactions were carried out on total RNA prepared from 0-hr L3,48-hr L3, 6-day L4, male and female adult worms using cuticlin-specificprimers. The results indicate that gene expression for both isoforms ofcuticlin were up-regulated prior to the molt, with maximal transcriptionat 48 hr-L3 and minimal expression at 0 hr and 6-day L4. There wasdetectable expression of both genes in male and female adult worms.

A homology search of a non-redundant protein database was performed withSEQ ID NO:4, using the BLASTX program available through the BLAST™network of the National Center for Biotechnology Information (NCBI)(National Library of Medicine, National Institutes of Health, Baltimore,Md.), available on the World Wide Web. This analysis showed thatDiCut-1A had significant homology to Ascaris cut-1 precursor at theamino acid level (bases 200 through 988 of SEQ ID NO:1 encode an aminoacid sequence that has 91% identity with the Ascaris cut-1 precursor),and nucleotides 434 through 880 of DiCut-1B encode an amino acidsequence that has 81% identity to the same Ascaris homolog.

Both Dirofilaria cuticlin cDNAs were expressed in a λ-cro expressionvector. DiCut-1A was expressed as a 44.5 kD histidine fusion protein,and DiCut-1B was expressed as a 31.1 kD fusion protein. Antibodiesraised to larval cuticles in rabbit and sera from a rabbit immunizedwith trickle doses of L3 stage larvae immunoreact with both forms ofcuticlin.

DiCut-1A and DiCut-1B cDNAs were used to probe Southern blots of genomicDNA from adult D. immitis. Both cDNAs hybridized to two almost identicalgenomic fragments suggesting that DiCut-1A and DiCut-1B are each encodedby a single copy gene. Because DiCut-1A and DiCut-1B are 75% identicalat the nucleotide level, it is likely that each cDNA may hybridize tothe other gene, as was seen in the present study. Interestingly, theDiCut-1A and DiCut-1B cDNA probes, were not cut internally by EcoRI, butdid hybridize to a number of fragments in genomic DNA digested withEcoRI. These results suggest the presence of introns within the cuticlingenes.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

1-11. (canceled)
 12. An isolated protein encoded by a nucleic acidmolecule that hybridizes in a solution comprising 2×SSC and 0%formamide, at a temperature of 37° C., and washing in 1×SSC and 0%formamide at a temperature of 64° C., to a polynucleotide moleculeconsisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:10.13. The protein of claim 12, wherein said protein comprises an aminoacid sequence at least 95% identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:4 and SEQ ID NO:9.
 14. Theprotein of claim 12, wherein said protein is encoded by a nucleic acidmolecule comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:10.15-16. (canceled)
 17. A method to identify a compound capable ofinhibiting filariid cuticlin activity, said method comprising contactingan isolated Dirofilaria immitis cuticlin protein as set forth in claim12, with a putative inhibitory compound under conditions in which, inthe absence of said compound, said protein has cuticlin activity, anddetermining if said putative inhibitory compound inhibits said activity.18-20. (canceled)
 21. The isolated protein of claim 12, wherein saidprotein elicits an immune response against a protein consisting of SEQID NO:4 or SEQ ID NO:9.
 22. The isolated protein of claim 12, whereinsaid nucleic acid molecule comprises a nucleic acid sequence at least95% identical to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:10.23. An isolated protein encoded by the nucleic acid molecule generatedby the polymerase chain reaction when said reaction is performed understandard conditions using: (a) Dirofilaria immitis larval cDNA as thetemplate; and (b) at least one nucleic acid primer comprising a sequenceselected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13 and SEQ ID NO:15, and wherein said nucleic acid molecule is atleast 1000 nucleotides in length.